Fucoidan nanogels and methods of their use and manufacture

ABSTRACT

Described herein are polymeric drug-carrying nanogels that are capable of targeting to P-selectin for the treatment of cancer and other diseases and conditions associated with P-selectin. Furthermore, in certain embodiments, the nanogels presented here offer a drug release mechanism based on acidic pH in the microenvironment of a tumor, thereby providing improved treatment targeting capability and allowing use of lower drug doses, thereby reducing toxicity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/689,683, filed Apr. 17, 2015, which claims the benefit of andpriority to U.S. Provisional Patent Application No. 61/980,643, filed onApr. 17, 2014, the entire contents of both of which are incorporatedherein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberDP2-HD075698 awarded by NIH. The government has certain rights in theinvention.

TECHNICAL FIELD

This invention relates generally to nanogels and methods of theirmanufacture and therapeutic use. In particular embodiments, theinvention relates to polymeric fucoidan-based nanogel vehicles for thetreatment of cancer and other diseases associated with P-selectin.

BACKGROUND

Nanogels—porous nanoscale hydrogel networks—are a class of nanomaterialswith tunable chemical properties that facilitate targeting and deliveryto specific tissues. They are intrinsically porous and can be loadedwith small drugs or macromolecules by physical entrapment, covalentconjugation or controlled self-assembly. The porosity of nanogelsprotect the drugs they carry from degradation and environmental hazards;hence, nanogels can be used as drug delivery agents and contrast agentsfor medical imaging.

Fucoidans are a class of sulfated, fucose-rich polymers that can befound, for example, in brown macroalgae. Fucoidans have been reported tohave anticoagulant, antiviral, anti-inflammatory, and anticanceractivities, as well as high affinity to P-selectin. P-selectin is aninflammatory cell adhesion molecule responsible for leukocyterecruitment and platelet binding. It is expressed constitutively inendothelial cells where it is stored in intracellular granules(Weibel-Palade bodies). Upon endothelial activation with endogenouscytokines or exogenous stimuli such as ionizing radiation, P-selectintranslocates to the cell membrane and into the lumen of blood vessels.P-selectin expression has been found to increase significantly in thevasculature of human lung, breast, and kidney cancers. P-selectin hasbeen shown to facilitate the process of metastasis by coordinating theinteraction between cancer cells, activated platelets and activatedendothelial cells.

It has been unexpectedly found that P-selectin is expressed in stromaand cancer cells in may human tumors, as well as in vasculature. Onlyone previous report describes P-selectin expression in cancer cells—ametastatic pancreatic tumor cell line. The phenomenon of tumor cellexpression of endothelial-specific adhesion molecules such as ICAM-1,VCAM-1, CD31/PECAM-1 and VE-cadherin has been applied to various typesof cancer cells and associated with increased metastasis and poorpatient prognosis.

The direct administration of fucoidan as a treatment for tumors ormetastases can be ineffective, due to toxicity limitations and lack ofdrug targeting. Disseminated tumors are poorly accessible to nanoscaledrug delivery systems due to the vascular barrier, which preventssufficient extravasation at the tumor site. Strategies to target leakyvasculature via the enhanced permeability and retention (EPR) effecthave shown little efficacy on avascular tumors and small metastases.

The clinical potential of nanomedicines has not yet been fulfilled inpart due to the endothelium barrier which limits the extravasation ofnanoparticles from the circulation into solid tumors. Passive targetingmechanisms such as the enhanced permeability and retention “EPR” effectshow preclinical efficacy. Yet the effect is less effective in smalltumors and metastases. Endothelial cells (EC) in the neovasculature arepromising targets due to their genetic stability and exposure to thecirculation. Nanoparticle drug carriers targeting the neovasculature arecurrently under clinical development, however, targeted delivery oftherapeutic agents to micro-metastases or tumors lacking neovasculatureremains an enduring challenge.

A nanogel containing fucoidan has been produced by chemical acetylationof the hydroxyl groups of fucoidan, rendering it amphipilic and able toform nanoparticles loaded with doxorubicin (Lee et al., CarbohydratePolymers 95 (2013) 606-614). However, by acetylating the hydroxyl groupsof fucose, specific affinity of the drug-containing nanogel toP-selectin is eliminated, thereby adversely affecting the ability of thenanogel to target cancer and other diseases associated with P-selectin.

There exists a need for a fucoidan-based nanogel that has a specificaffinity to P-selectin to treat cancer and other diseases and conditionsassociated with P-selectin.

SUMMARY OF THE INVENTION

Described herein are polymeric drug-carrying nanogels that are capableof targeting to P-selectin and, therefore, are useful in the treatmentof cancer and other diseases and conditions associated with P-selectin.Without wishing to be bound to any particular theory, specific affinityto P-selectin requires both free hydroxyls and a proximate negativecharge. Thus, presented herein are nanogels having hydroxyls andsulfates that are free for targeting to P-selectin. Furthermore, incertain embodiments, the nanogels presented here offer a drug releasemechanism based on acidic pH in the microenvironment of a tumor, therebyproviding improved treatment targeting capability and allowing use oflower drug doses, thereby reducing toxicity.

P-selectin is a new target for drug delivery in various cancers andcontributes both at the tissue level and the cellular level. SinceP-selectin is highly involved in inflammatory processes, it is usefulfor inflammatory diseases such as arthritis and atherosclerosis, whichalso involve P-selectin on endothelial cells. P-selectin is a celladhesion molecule known to facilitate metastasis which is expressed inthe vasculature of many human tumors. A delivery nanoparticle platformwas developed using an algae-derived polysaccharide with intrinsicnanomolar affinity to P-selectin. The nanoparticles target primary andmetastatic tumors to impart a significant anti-tumor activity comparedto untargeted nanoparticles encapsulating chemotherapies. Single-doseadministration of an encapsulated reversible MEK inhibitor results inprolonged inhibition of ERK phosphorylation and increased apoptosis atthe tumor site. Additionally, ionizing radiation-induced P-selectinexpression guides the targeted nanoparticles to the tumor site,demonstrating a potential strategy to target disparate drug classes toalmost any tumor.

P-selectin was identified as a useful target for drug delivery and wasused in a set of in vivo and in vitro models to explore its anti-tumoreffectiveness, with multiple applications such as targeting aggressiveprimary and metastatic tumors using irreversible chemotherapies andreversible kinase inhibitor.

In certain embodiments, the nanogels described herein present fucoidanon their surface, specifically targeting P-selectin on activatedplatelets and activated endothelium. The fucoidan on the surface of thenanoparticles making up the nanogel have free hydroxyl moieties and freesulfate moieties. The nanoparticles release the drug moieties theycontain in the acidic tumor microenvironment and lysosomes. The fucoidanalso appears to act as an immunomodulator, likely inducing an immuneresponse against the tumor.

In a specific embodiment, a fucoidan-based nanogel is presented thatdelivers doxorubicin and releases it via pH-sensitive degradation of ahydrazone bond. The doxorubicin is chemically conjugated to polyethyleneglycol (PEG), but is only electrostatically bound to the anionic polymerfucoidan. In other embodiments, other cationic drugs may be used, forexample, vincristine. The particle size and charge can be modifiedaccording to the intended use.

In other specific embodiments described herein, nanogels are synthesizedby non-covalent assembly of fucoidan with a hydrophobic drug.Nanoparticle-drug assemblies synthesized using this method include, forexample, particles encapsulating one or more of paclitaxel, MEK162, andispinesib.

Also, in certain embodiments, the invention encompasses methods oftreatment of disease associated with P-selectin using the compositionsdescribed herein. For example, the compositions may be used in thetreatment of malignant neoplasms including carcinomas, sarcomas,lymphomas, and leukemia. Furthermore, the compositions may be used inother P-selectin-associated diseases such as sickle cell disease,arterial thrombosis, rheumatoid arthritis, ischemia, and reperfusion.Combination therapies are contemplated herein. Also, the use ofcompositions described herein with radiotherapy for improved P-selectintargeting and activity is contemplated.

In one aspect, the invention is directed to a polymeric nanogel withaffinity to P-selectin, the nanogel comprises: (i) a sulfated polymerspecies comprising free hydroxyl moieties and sulfate moieties capableof targeting to P-selectin; and (ii) a drug.

In certain embodiments, the sulfated polymer species is a sulfatedpolysaccharide and/or protein. In certain embodiments, the drug is acationic drug.

In certain embodiments, the sulfated polymer species is a fucoidan. Incertain embodiments, the fucoidan is a sulfated polysaccharidecomprising sulfated ester moieties of fucose.

In certain embodiments, the nanogel comprises nanoparticles that have acore comprising albumin and a surface comprising fucoidan. In certainembodiments, the nanogel comprises polyethylene glycol (PEG), whereinthe drug is conjugated to the polyethylene glycol via hydrozonelinkages.

In certain embodiments, the drug is not chemically conjugated to thesulfated polymer species, but is electrostatically bound to the sulfatedpolymer species. In certain embodiments, the sulfated polymer species isa fucoidan.

In certain embodiments, the drug is doxorubicin (DOX){(7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione}(trade name Adriamycin).

In certain embodiments, the drug is vincristine{(3aR,3a1R,4R,5S,5aR,10bR)-methyl4-acetoxy-3a-ethyl-9-((5S,7S,9S)-5-ethyl-5-hydroxy-9-(methoxycarbonyl)-2,4,5,6,7,8,9,10-octahydro-1H-3,7-methano[1]azacycloundecino[5,4-b]indol-9-yl)-6-formyl-5-hydroxy-8-methoxy-3a,3a1,4,5,5a,6,11,12-octahydro-1H-indolizino[8,1-cd]carbazole-5-carboxylate}.

In certain embodiments, the cationic drug comprises one or more membersselected from the group consisting of: DOX, vincristine,paclitaxel{(2α,4α,5β,7β,10β,13α)-4,10-bis(acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-ylbenzoate}, MEK162{6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide},ispinesib{N-(3-aminopropyl)-N-[(1R)-1-(3-benzyl-7-chloro-4-oxoquinazolin-2-yl)-2-methylpropyl]-4-methylbenzamide},daunorubicin (daunomycin){(8S,10S)-8-acetyl-10-[(2S,4S,5S,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,8,11-trihydroxy-1-methoxy-9,10-dihydro-7H-tetracene-5,12-dione},epirubicin{(8R,10S)-10-((2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione},idarubicin{(1S,3S)-3-acetyl-3,5,12-trihydroxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl3-amino-2,3,6-trideoxo-α-L-lyxo-hexopyranoside}, valrubicin{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-4-({2,3,6-trideoxy-3-[(trifluoroacetyl)amino]hexopyranosyl}oxy)-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethylpentanoate}, mitoxantrone {1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino)ethylamino]-anthracene-9,10-dione}, vinblastine {dimethyl(2β,3β,4β,5α,12β,19α)-15-[(5S,9S)-5-ethyl-5-hydroxy-9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methanoazacycloundecino[5,4-b]indol-9-yl]-3-hydroxy-16-methoxy-1-methyl-6,7-didehydroaspidospermidine-3,4-dicarboxylate},vindesine {methyl(5S,7S,9S)-9-[(2β,3β,4β,5a,12β,19α)-3-(aminocarbonyl)-3,4-dihydroxy-16-methoxy-1-methyl-6,7-didehydroaspidospermidin-15-yl]-5-ethyl-5-hydroxy-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methanoazacycloundecino[5,4-b]indole-9-carboxylate},vinorelbine{4-(acetyloxy)-6,7-didehydro-15-((2R,6R,8S)-4-ethyl-1,3,6,7,8,9-hexahydro-8-(methoxycarbonyl)-2,6-methano-2H-azecino(4,3-b)indol-8-yl)-3-hydroxy-16-methoxy-1-methyl-methylester}, bleomycin{(3-{[(2′-{(5S,8S,9S,10R,13S)-15-{6-amino-2-[(1S)-3-amino-1-{[(2S)-2,3-diamino-3-oxopropyl]amino-3-oxopropyl]-5-methylpyrimidin-4-yl}-13-[{[(2R,3S,4S,5S,6S)-3-{[(2R,3S,4S,5R,6R)-4-(carbamoyloxy)-3,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]oxy}-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]oxy}(1H-imidazol-5-yl)methyl]-9-hydroxy-5-[(1R)-1-hydroxyethyl]-8,10-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazapentadec-1-yl}-2,4′-bi-1,3-thiazol-4-yl)carbonyl]aminopropyl)(dimethyl)sulfonium}, actinomycin D (dactinomycin){2-Amino-N,N′-bis[(6S,9R,10S,13R,18aS)-6,13-diisopropyl-2,5,9-trimethyl-1,4,7,11,14-pentaoxohexadecahydro-1H-pyrrolo[2,1-1][1,4,7,10,13]oxatetraazacyclohexadecin-10-yl]-4,6-dimethyl-3-oxo-3H-phenoxazine-1,9-dicarboxamide},sorafenib{4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide},camptothecin{(S)-4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione},topotecan{(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dionemonohydrochloride}, and irinotecan{(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4′bipiperidine]-1′-carboxylate}.

In certain embodiments, the nanogel comprises fucoidan and DOX-PEG-DOXconstructs. In certain embodiments, the nanogel comprises fucoidan onthe surface of nanoparticles of the nanogel. In certain embodiments, thenanogel comprises particles having an average particle diameter of fromabout 20 nm to about 400 nm (e.g., from about 100 nm to about 200 nm, orfrom about 150 nm to about 170 nm).

In certain embodiments, the nanogel further comprises a fluorophore. Incertain embodiments, the fluorophore is a near infra-red dye. In certainembodiments, the near infra-red dye is IR783{2-[2-[2-Chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indoliumhydroxide, inner salt sodium salt}.

In certain embodiments, the nanogel is a pharmaceutical composition. Incertain embodiments, the nanogel is pharmaceutically acceptable.

In another aspect, the invention is directed to a method of treating aP-selectin associated disease, the method comprising a step ofadministering to a subject in need of treatment a formulation comprisinga polymeric nanogel with affinity to P-selectin, the nanogel comprising:(i) a sulfated polymer species comprising free hydroxyl moieties andsulfate moieties capable of targeting to P-selectin; and (ii) a drug;wherein the nanogel binds to P-selectin and translocates an activeendothelial barrier. In certain embodiments, the sulfated polymerspecies is a sulfated polysaccharide and/or protein. In certainembodiments, the drug is a cationic drug.

In certain embodiments, the subject is human. In certain embodiments,the formulation is a therapeutic agent.

In certain embodiments, the P-selectin associated disease is a memberselected from the group consisting of carcinoma, sarcoma, lymphoma,leukemia, sickle cell disease, arterial thrombosis, rheumatoidarthritis, ischemia, and reperfusion.

In certain embodiments, the method comprises administering aradiotherapeutic, wherein the nanogel provides improved P-selectintargeting and activity.

In certain embodiments, the step of administering the nanogel results intargeted delivery of the drug to P-selectin. In certain embodiments,upon delivery of the drug to P-selectin, a local environment having anacidic pH causes release of the drug from the nanogel. In certainembodiments, the nanogel comprises PEG and the local acidic pHenvironment results in breakage of hydrozone linkages between the PEGand the drug.

In another aspect, the invention is directed to a method formanufacturing a nanogel comprising contacting fucoidan and a drug-PEGconstruct in the presence of a salt to form hydrogel aggregates, andagitating the hydrogel aggregates to form nanoparticles.

In certain embodiments, the drug-PEG construct is DOX-PEG-DOX. Incertain embodiments, the salt is a phosphonobile salt (PBS). In certainembodiments, the agitating includes sonicating the hydrogel aggregates.

In another aspect, the invention is directed to a method formanufacturing a nanogel comprising contacting albumin, fucoidan, andsorafenib in an aqueous salt solution to form hydrogel aggregates, andagitating the hydrogel aggregates to form nanoparticles. In certainembodiments, the albumin is Human Serum Albumin. In certain embodiments,the salt solution is a phosphonobile salt (PBS). In certain embodiments,agitating includes sonicating the hydrogel aggregates.

In another aspect, the invention is directed to a method formanufacturing a nanogel comprising contacting fucoidan and paclitaxel inan aqueous solution to form hydrogel aggregates, and agitating thehydrogel aggregates to form nanoparticles.

In certain embodiments, agitating includes sonicating the hydrogelaggregates.

In another aspect, the invention is directed to a polymericfucoidan-based nanogel with affinity to P-selectin, the nanogelcomprising a non-covalent assembly of fucoidan and a hydrophobic drug.

In certain embodiments, the hydrophobic drug comprises one or moremembers selected from the group consisting ofpaclitaxel{(2α,4α,5β,7β,10β,13α)-4,10-bis(acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-ylbenzoate}, docetaxel{1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-triyl4-acetate 2-benzoate13-{(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropanoate}},Camptothecin{(S)-4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione},MEK162{6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide},sorafenib{4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide},ispinesib{N-(3-aminopropyl)-N-[(1R)-1-(3-benzyl-7-chloro-4-oxoquinazolin-2-yl)-2-methylpropyl]-4-methylbenzamide},LY294002 {2-Morpholin-4-yl-8-phenylchromen-4-one}, Selumetinib{6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide},PD184352{2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide},5-fluorouracil {5-fluoro-1H,3H-pyrimidine-2,4-dione}, Cyclophosphamide{(RS)—N,N-bis(2-chloroethyl)-1,3,2-oxazaphosphinan-2-amine 2-oxide},Atorvastatin{(3R,5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoicacid}, Lovastatin{(1S,3R,7S,8S,8aR)-8-{2-[(2R,4R)-4-hydroxy-6-oxooxan-2-yl]ethyl}-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl(2S)-2-methylbutanoate}, etoposide {4′-Demethyl-epipodophyllotoxin9-[4,6-O—(R)-ethylidene-beta-D-glucopyranoside], 4′-(dihydrogenphosphate)}, dexamethasone{(8S,9R,10S,11S,13S,14S,16R,17R)-9-Fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one},gemcitabine{4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranosyl)pyrimidin-2(1H)-on},Rapamycin (Sirolimus){(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone}, and methotrexate{(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}benzoyl)amino]pentanedioicacid}.

Other features, objects, and advantages of the present invention areapparent in the detailed description and claims that follow. It shouldbe understood, however, that the detailed description and claims, whileindicating embodiments of the present invention, are given by way ofillustration only, not limitation. Various changes and modificationswithin the scope of the invention will become apparent to those skilledin the art.

Definitions

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Administration”: The term “administration” refers to introducing asubstance into a subject. In general, any route of administration may beutilized including, for example, parenteral (e.g., intravenous), oral,topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal,rectal, nasal, introduction into the cerebrospinal fluid, orinstillation into body compartments. In some embodiments, administrationis oral. Additionally or alternatively, in some embodiments,administration is parenteral. In some embodiments, administration isintravenous.

“Amino Acid”: As used herein, the term “amino acid,” in its broadestsense, refers to any compound and/or substance that can be incorporatedinto a polypeptide chain. In some embodiments, an amino acid has thegeneral structure H2N—C(H)(R)—COOH. In some embodiments, an amino acidis a naturally occurring amino acid. In some embodiments, an amino acidis a synthetic amino acid; in some embodiments, an amino acid isad-amino acid; in some embodiments, an amino acid is an 1-amino acid.“Standard amino acid” refers to any of the twenty standard 1-amino acidscommonly found in naturally occurring peptides. “Nonstandard amino acid”refers to any amino acid, other than the standard amino acids,regardless of whether it is prepared synthetically or obtained from anatural source. As used herein, “synthetic amino acid” encompasseschemically modified amino acids, including but not limited to salts,amino acid derivatives (such as amides), and/or substitutions. Aminoacids, including carboxy- and/or amino-terminal amino acids in peptides,can be modified by methylation, amidation, acetylation, protectinggroups, and/or substitution with other chemical groups that can changethe peptide's circulating half-life without adversely affecting theiractivity. Amino acids may participate in a disulfide bond. Amino acidsmay comprise one or posttranslational modifications, such as associationwith one or more chemical entities (e.g., methyl groups, acetate groups,acetyl groups, phosphate groups, formyl moieties, isoprenoid groups,sulfate groups, polyethylene glycol moieties, lipid moieties,carbohydrate moieties, biotin moieties, etc.). The term “amino acid” isused interchangeably with “amino acid residue,” and may refer to a freeamino acid and/or to an amino acid residue of a peptide. It will beapparent from the context in which the term is used whether it refers toa free amino acid or a residue of a peptide.

“Antibody polypeptide”: As used herein, the terms “antibody polypeptide”or “antibody”, or “antigen-binding fragment thereof’, which may be usedinterchangeably, refer to polypeptide(s) capable of binding to anepitope. In some embodiments, an antibody polypeptide is a full-lengthantibody, and in some embodiments, is less than full length but includesat least one binding site (comprising at least one, and preferably atleast two sequences with structure of antibody “variable regions”). Insome embodiments, the term “antibody polypeptide” encompasses anyprotein having a binding domain which is homologous or largelyhomologous to an immunoglobulin-binding domain. In particularembodiments, “antibody polypeptides” encompasses polypeptides having abinding domain that shows at least 99% identity with animmunoglobulinbinding domain. In some embodiments, “antibody polypeptide” is anyprotein having a binding domain that shows at least 70%, 80%, 85%, 90%,or 95% identity with an immunoglobulin binding domain, for example areference immunoglobulin binding domain. An included “antibodypolypeptide” may have an amino acid sequence identical to that of anantibody that is found in a natural source. Antibody polypeptides inaccordance with the present invention may be prepared by any availablemeans including, for example, isolation from a natural source orantibody library, recombinant production in or with a host system,chemical synthesis, etc., or combinations thereof. An antibodypolypeptide may be monoclonal or polyclonal. An antibody polypeptide maybe a member of any immunoglobulin class, including any of the humanclasses: IgG, IgM, IgA, IgD, and IgE. In certain embodiments, anantibody may be a member of the IgG immunoglobulin class. As usedherein, the terms “antibody polypeptide” or “characteristic portion ofan antibody” are used interchangeably and refer to any derivative of anantibody that possesses the ability to bind to an epitope of interest.In certain embodiments, the “antibody polypeptide” is an antibodyfragment that retains at least a significant portion of the full-lengthantibody's specific binding ability. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFvdiabody, and Fd fragments. Alternatively or additionally, an antibodyfragment may comprise multiple chains that are linked together, forexample, by disulfide linkages. In some embodiments, an antibodypolypeptide may be a human antibody. In some embodiments, the antibodypolypeptides may be a humanized. Humanized antibody polypeptides includemay be chimeric immunoglobulins, immunoglobulin chains or antibodypolypeptides (such as Fv, Fab, Fab′, F(ab′)2 or other antigen bindingsubsequences of antibodies) that contain minimal sequence derived fromnon-human immunoglobulin. In general, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary-determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity.

“Antigen”: As used herein, the term “antigen” is a molecule or entity towhich an antibody binds. In some embodiments, an antigen is or comprisesa polypeptide or portion thereof. In some embodiments, an antigen is aportion of an infectious agent that is recognized by antibodies. In someembodiments, an antigen is an agent that elicits an immune response;and/or (ii) an agent that is bound by a T cell receptor (e.g., whenpresented by an MHC molecule) or to an antibody (e.g., produced by a Bcell) when exposed or administered to an organism. In some embodiments,an antigen elicits a humoral response (e.g., including production ofantigen-specific antibodies) in an organism; alternatively oradditionally, in some embodiments, an antigen elicits a cellularresponse (e.g., involving T-cells whose receptors specifically interactwith the antigen) in an organism. It will be appreciated by thoseskilled in the art that a particular antigen may elicit an immuneresponse in one or several members of a target organism (e.g., mice,rabbits, primates, humans), but not in all members of the targetorganism species. In some embodiments, an antigen elicits an immuneresponse in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ofthe members of a target organism species. In some embodiments, anantigen binds to an antibody and/or T cell receptor, and may or may notinduce a particular physiological response in an organism. In someembodiments, for example, an antigen may bind to an antibody and/or to aT cell receptor in vitro, whether or not such an interaction occurs invivo. In general, an antigen may be or include any chemical entity suchas, for example, a small molecule, a nucleic acid, a polypeptide, acarbohydrate, a lipid, a polymer[in some embodiments other than abiologic polymer (e.g., other than a nucleic acid or amino acidpolymer)] etc. In some embodiments, an antigen is or comprises apolypeptide. In some embodiments, an antigen is or comprises a glycan.Those of ordinary skill in the art will appreciate that, in general, anantigen may be provided in isolated or pure form, or alternatively maybe provided in crude form (e.g., together with other materials, forexample in an extract such as a cellular extract or other relativelycrude preparation of an antigen-containing source). In some embodiments,antigens utilized in accordance with the present invention are providedin a crude form. In some embodiments, an antigen is or comprises arecombinant antigen.

“Associated”: As used herein, the term “associated” typically refers totwo or more entities in physical proximity with one another, eitherdirectly or indirectly (e.g., via one or more additional entities thatserve as a linking agent), to form a structure that is sufficientlystable so that the entities remain in physical proximity under relevantconditions, e.g., physiological conditions. In some embodiments,associated moieties are covalently linked to one another. In someembodiments, associated entities are non-covalently linked. In someembodiments, associated entities are linked to one another by specificnon-covalent interactions (i.e., by interactions between interactingligands that discriminate between their interaction partner and otherentities present in the context of use, such as, for example,streptavidin/avidin interactions, antibody/antigen interactions, etc.).Alternatively or additionally, a sufficient number of weakernon-covalent interactions can provide sufficient stability for moietiesto remain associated. Exemplary non-covalent interactions include, butare not limited to, electrostatic interactions, hydrogen bonding,affinity, metal coordination, physical adsorption, host-guestinteractions, hydrophobic interactions, pi stacking interactions, vander Waals interactions, magnetic interactions, electrostaticinteractions, dipole-dipole interactions, etc.

As used herein, for example, within the claims, the term “ligand”encompasses moieties that are associated with another entity, such as ananogel polymer, for example. Thus, a ligand of a nanogel polymer can bechemically bound to, physically attached to, or physically entrappedwithin, the nanogel polymer, for example.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe materials that do not elicit a substantial detrimental responsein vivo. In certain embodiments, the materials are “biocompatible” ifthey are not toxic to cells. In certain embodiments, materials are“biocompatible” if their addition to cells in vitro results in less thanor equal to 20% cell death, and/or their administration in vivo does notinduce inflammation or other such adverse effects. In certainembodiments, materials are biodegradable.

“Biodegradable”: As used herein, “biodegradable” materials are thosethat, when introduced into cells, are broken down by cellular machinery(e.g., enzymatic degradation) or by hydrolysis into components thatcells can either reuse or dispose of without significant toxic effectson the cells. In certain embodiments, components generated by breakdownof a biodegradable material do not induce inflammation and/or otheradverse effects in vivo. In some embodiments, biodegradable materialsare enzymatically broken down. Alternatively or additionally, in someembodiments, biodegradable materials are broken down by hydrolysis. Insome embodiments, biodegradable polymeric materials break down intotheir component polymers. In some embodiments, breakdown ofbiodegradable materials (including, for example, biodegradable polymericmaterials) includes hydrolysis of ester bonds. In some embodiments,breakdown of materials (including, for example, biodegradable polymericmaterials) includes cleavage of urethane linkages.

“Carrier”: As used herein, “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the compound is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water or aqueous solution saline solutions and aqueous dextrose andglycerol solutions are preferably employed as carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. In someembodiments, the composition described herein is a carrier.

“Combination Therapy”: As used herein, the term “combination therapy”,refers to those situations in which two or more different pharmaceuticalagents for the treatment of disease are administered in overlappingregimens so that the subject is simultaneously exposed to at least twoagents. In some embodiments, the different agents are administeredsimultaneously. In some embodiments, the administration of one agentoverlaps the administration of at least one other agent. In someembodiments, the different agents are administered sequentially suchthat the agents have simultaneous biologically activity with in asubject.

“Hydrolytically degradable”: As used herein, “hydrolytically degradable”materials are those that degrade by hydrolytic cleavage. In someembodiments, hydrolytically degradable materials degrade in water. Insome embodiments, hydrolytically degradable materials degrade in waterin the absence of any other agents or materials. In some embodiments,hydrolytically degradable materials degrade completely by hydrolyticcleavage, e.g., in water. By contrast, the term “non-hydrolyticallydegradable” typically refers to materials that do not fully degrade byhydrolytic cleavage and/or in the presence of water (e.g., in the solepresence of water).

“Pharmaceutically acceptable”: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

“Pharmaceutical composition”: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In some embodiments, activeagent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probabilityof achieving a predetermined therapeutic effect when administered to arelevant population. In some embodiments, pharmaceutical compositionsmay be specially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

“Protein”: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least 3-5 amino acids linked to one another bypeptide bonds). Proteins may include moieties other than amino acids(e.g., may be glycoproteins, proteoglycans, etc.) and/or may beotherwise processed or modified. In some embodiments “protein” can be acomplete polypeptide as produced by and/or active in a cell (with orwithout a signal sequence); in some embodiments, a “protein” is orcomprises a characteristic portion such as a polypeptide as produced byand/or active in a cell. In some embodiments, a protein includes morethan one polypeptide chain. For example, polypeptide chains may belinked by one or more disulfide bonds or associated by other means. Insome embodiments, proteins or polypeptides as described herein maycontain L amino acids, D-amino acids, or both, and/or may contain any ofa variety of amino acid modifications or analogs known in the art.Useful modifications include, e.g., terminal acetylation, amidation,methylation, etc. In some embodiments, proteins or polypeptides maycomprise natural amino acids, non-natural amino acids, synthetic aminoacids, and/or combinations thereof. In some embodiments, proteins are orcomprise antibodies, antibody polypeptides, antibody fragments,biologically active portions thereof, and/or characteristic portionsthereof.

“Physiological conditions”: The phrase “physiological conditions”, asused herein, relates to the range of chemical (e.g., pH, ionic strength)and biochemical (e.g., enzyme concentrations) conditions likely to beencountered in the intracellular and extracellular fluids of tissues.For most tissues, the physiological pH ranges from about 7.0 to 7.4.

“Polypeptide”: The term “polypeptide” as used herein, refers to a stringof at least three amino acids linked together by peptide bonds. In someembodiments, a polypeptide comprises naturally-occurring amino acids;alternatively or additionally, in some embodiments, a polypeptidecomprises one or more non-natural amino acids (i.e., compounds that donot occur in nature but that can be incorporated into a polypeptidechain; see, for example,http://www.cco.caltech.edu/^(˜)dadgrp/Unnatstruct.gif, which displaysstructures of non-natural amino acids that have been successfullyincorporated into functional ion channels) and/or amino acid analogs asare known in the art may alternatively be employed). In someembodiments, one or more of the amino acids in a protein may bemodified, for example, by the addition of a chemical entity such as acarbohydrate group, a phosphate group, a farnesyl group, an isofarnesylgroup, a fatty acid group, a linker for conjugation, functionalization,or other modification, etc.

“Polysaccharide”: The term “polysaccharide” refers to a polymer ofsugars. Typically, a polysaccharide comprises at least three sugars. Insome embodiments, a polypeptide comprises natural sugars (e.g., glucose,fructose, galactose, mannose, arabinose, ribose, and xylose);alternatively or additionally, in some embodiments, a polypeptidecomprises one or more non-natural amino acids (e.g, modified sugars suchas 2′-fluororibose, 2′-deoxyribose, and hexose).

“Substantially”: As used herein, the term “substantially”, and grammaticequivalents, refer to the qualitative condition of exhibiting total ornear-total extent or degree of a characteristic or property of interest.One of ordinary skill in the art will understand that biological andchemical phenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result.

“Subject”: As used herein, the term “subject” includes humans andmammals (e.g., mice, rats, pigs, cats, dogs, and horses). In manyembodiments, subjects are be mammals, particularly primates, especiallyhumans. In some embodiments, subjects are livestock such as cattle,sheep, goats, cows, swine, and the like; poultry such as chickens,ducks, geese, turkeys, and the like; and domesticated animalsparticularly pets such as dogs and cats. In some embodiments (e.g.,particularly in research contexts) subject mammals will be, for example,rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine suchas inbred pigs and the like.

“Therapeutic agent”: As used herein, the phrase “therapeutic agent”refers to any agent that has a therapeutic effect and/or elicits adesired biological and/or pharmacological effect, when administered to asubject.

“Treatment”: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance that partiallyor completely alleviates, ameliorates, relives, inhibits, delays onsetof, reduces severity of, and/or reduces incidence of one or moresymptoms, features, and/or causes of a particular disease, disorder,and/or condition. Such treatment may be of a subject who does notexhibit signs of the relevant disease, disorder and/or condition and/orof a subject who exhibits only early signs of the disease, disorder,and/or condition. Alternatively or additionally, such treatment may beof a subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In some embodiments, treatment maybe of a subject who has been diagnosed as suffering from the relevantdisease, disorder, and/or condition. In some embodiments, treatment maybe of a subject known to have one or more susceptibility factors thatare statistically correlated with increased risk of development of therelevant disease, disorder, and/or condition.

Drawings are presented herein for illustration purposes only, not forlimitation.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects, features, and advantages ofthe present disclosure will become more apparent and better understoodby referring to the following description taken in conjunction with theaccompanying drawings, in which:

FIG. 1 is a schematic diagram illustrating the preparation ofpH-sensitive fucoidan nanogels for the delivery of doxorubicin (FiDOX),according to an illustrative embodiment of the invention.

FIG. 2A shows vials containing fucoidan-paclitaxel nanoparticles(FiPXL), according to an illustrative embodiment of the invention.

FIG. 2B shows the chemical structure of paclitaxel (PX) and fucoidan(Fi) in the fucoidan-paclitaxel nanoparticles, according to anillustrative embodiment of the invention.

FIG. 3A are plots of dynamic light scattering measurements of FiDOXnanogels, according to an illustrative embodiment of the invention.

FIG. 3B are transmission electron microscope images of nanogels,according to an illustrative embodiment of the invention.

FIG. 4 is a graph showing rate of release of doxorubicin from FiDOXnanogels over time, as a function of pH, according to an illustrativeembodiment of the invention.

FIG. 5A shows a plot of fluorescence intensity demonstrating in vitroactivity of FiDOX nanogels, according to an illustrative embodiment ofthe invention.

FIG. 5B shows a plot of an MTT cell viability assay, according to anillustrative embodiment of the invention.

FIG. 6 shows bioluminescence images demonstrating anti-tumor efficacy ofFiDOX nanogels, according to an illustrative embodiment of theinvention.

FIG. 7 is a plot of bioluminescence showing anti-tumor efficacy of FiDOXnanogels, according to an illustrative embodiment of the invention.

FIG. 8 is a plot showing laboratory mouse survival curve data followinginjection of FiDOX nanogel, according to an illustrative embodiment ofthe invention.

FIG. 9 is a schematic showing fucoidan-ispinesib nanogels (Fi-ISP) andanalogous nanoparticles, according to an illustrative embodiment of theinvention.

FIGS. 10A and 10B are electron micrographs of fucoidan-ispenesibnanoparticles (Fi-ISP) and PGA-ispinesib nanoparticles, according to anillustrative embodiment of the invention.

FIG. 11 is an electron micrograph of fucoidan-MEK162 nanoparticles,according to an illustrative embodiment of the invention.

FIGS. 12A-12C illustrate P-selectin expression in human cancers.

FIG. 12A illustrates human tissue microarrays (TMA) stained withP-selectin antibody (Lymphoma normal tissue is from the spleen; Lymphoma1: non-Hodgkin B cell lymphoma (Lymph node); 2: peripheral T celllymphoma (Lymph node); 3: brain metastases of non-Hodgkin B celllymphoma; Lung cancer 1: lung squamous cell carcinoma; 2: small cellundifferentiated carcinoma; 3: metastatic lung adenocarcinoma; Breastcancer 1: Infiltrating ductal carcinoma; 2: advanced infiltrating ductalcarcinoma; 3: lymph node metastases of infiltrating ductal carcinoma.)

FIG. 12B illustrates a percentage of positively stained samples from theTMAs calculated with imaging software.

FIG. 12C illustrates data from The Cancer Genome Atlas showingP-selectin gene alterations in various cancers (red denotesamplification, green denotes mutation, blue denotes deletion, and graydenotes multiple alterations).

FIGS. 12D-12E illustrate a preparation scheme of P-selectin targetednanoparticles.

FIG. 12D illustrates preparation schemes for fucoidan-encapsulatedpaclitaxel nanoparticles (FiPAX) via nanoprecipitation (top) anddoxorubicin-encapsulated fucoidan nanoparticles (FiDOX) (bottom) vialayer-by-layer assembly, and SEM images of FiPAX and FiDOX nanoparticles(right).

FIG. 12E illustrates binding of IR783 dye loaded FiPAX to immobilizedhuman recombinant P-selectin after 15 min of incubation. Fluorescencewas measured with a fluorescent plate reader.

FIGS. 13A-13E illustrate anti-tumor efficacy of FiPAX vs. DexPAX withand without radiation.

FIGS. 14A-14E illustrate selective endothelial/tumor penetrationassessments in vitro.

FIG. 14A illustrates assay to test penetration of nanoparticles into anactivated endothelial monolayer barrier and infiltration into spheroidscomposed of tumor cells from a small cell lung cancer patient uponactivation with TNF-α.

FIG. 14B illustrates fluorescence of FiPAX or DexPAX nanoparticles inthe upper and lower chambers was measured with a fluorescence platereader at 780 nm (excitation) and 815 nm (emission) after 1 h ofincubation.

FIG. 14C illustrates the endothelial monolayer component of the chamberwas visualized to estimate nanoparticle internalization using afluorescent microscope equipped with a NIR sensitive XM10 Olympus CCDcamera, binding/internalization of FiPAX or control DexPAX nanoparticles(red) to a bEnd.3 endothelial cell monolayer (CellMask Green membranestain) upon activation with TNF-α.

FIG. 14D illustrates fluorescence images of nanoparticle penetrationinto tumor spheres upon endothelial activation.

FIG. 14E illustrates quantification of tumor sphere uptake from 6 imagesper condition using ImageJ.

FIGS. 15A-15F illustrate targeting P-selectin positive and negativetumors in-vivo.

FIG. 15A illustrates high expression of P-selectin in a PDX model ofsmall cell lung cancer (top), and fluorescence efficiency from IR783loaded FiPAX and DexPAX injected to tumor bearing mice and imaged withIVIS 24 h and 72 h after injection, n=4 (bottom).

FIG. 15B illustrates tumor growth inhibition of a P-selectin expressingsmall cell lung cancer PDX after a single treatment on day 12, n=10.

FIG. 15C illustrates radiation induced expression of P-selectin in micewith bilateral 3LL tumors treated with 6 Gy gamma radiation on the rightflank tumor only.

FIG. 15D illustrates a percentage of P-selectin positive blood vesselsfrom entire CD31 stained blood vessels. Data is presented as the mean of4 images per timepoint at 10×.

FIG. 15E illustrates fluorescence efficiency from IR783 loaded FiPAX andDexPAX injected to 3LL tumor bearing mice with or without treatment of 6Gy gamma radiation on the right flank tumor only.

FIG. 15F illustrates tumor growth inhibition via single administrationof nanoparticles after radiation treatment. The data is presented asmean±standard error.

FIG. 16 illustrates FiDOX efficacy in lung metastasis, P-selectinexpression, and Bio distribution of FiDOX.

FIGS. 17A-17E illustrate the efficacy of P-selectin targetednanoparticles in metastases.

FIG. 17A illustrates representative images of P-selectin and vasculature(CD31) staining in a B16F10 melanoma experimental lung metastasis model14 days after inoculation.

FIG. 17B illustrates survival data from two experiments using the B16F10metastasis model treated with a single injection on day 7 afterinoculation.

FIG. 17C illustrates survival data from two experiments using the B16F10metastasis model treated with a single injection on day 7 afterinoculation.

FIG. 17D illustrates bioluminescence images acquired 7 days after asingle administration of treatment with FiDOX, free doxorubicin (DOX),fucoidan vehicle (Fi), or PBS control.

FIG. 17E illustrates median photon count of the 6 treatment groupsmeasured by IVIS and quantified by LivingImage software.

FIGS. 18A-18E illustrate FiMEK improved pERK inhibition and efficacy.

FIGS. 19A-19D illustrate inhibition of MEK improved anti-tumor efficacyand induced apoptosis by P-selectin targeted nanoparticles in vitro andin vivo.

FIG. 19A illustrates proliferation of and A549 cell lines measured after4 days of treatment with MEK162 or FI-MEK as indicated (top), andbiochemical analysis of A375 and A549 cell lines treated for 4 hourswith MEK163 or FI-MEK (bottom).

FIG. 19B illustrates tumor growth of xenograft derived from A375 andSW620 treated once with vehicle, MEK162, FI-MEK or a daily dose ofMEK162 (n=6).

FIG. 19C illustrates biochemical (western blot) quantification of pERKand Cleavage PARP on xenografts A375 tumors treated for 2 and 16 hourswith MEK163 or FI-MEK.

FIG. 19D illustrates immunohistochemistry of Clevage PARP on xenogfratHCT116 tumors treated with MEK162 or MEK-IR.

FIG. 20A shows the size distribution of FiDOX, DexDOX, FiPAX, and DexPAXnanoparticles.

FIG. 20B shows the zeta potential of FiDOX, DexDOX, FiPAX, and DexPAXnanoparticles.

FIG. 20C shows SEM images of FiPAX and FiDOX nanoparticles. Scale bar is100 nm.

FIG. 20D shows that the sizes of FiDOX and FiPAX stays constant over a 5day period.

FIG. 20E shows the release of DOX over time for pH 7.4 and pH 5.5.

FIG. 20F shows release of PXL over time for pH 7.4 and pH 5.5.

FIG. 21 shows proliferation of cell lines was measured after 4 days oftreatment with MEK162 or FI-MEK as indicated. Blue-MEK162, RED-FiMEK.

FIG. 22 shows the drug release profile MEK162 drug from nanoparticlesover time at different pH.

FIG. 23A shows IHC staining of P-selectin expression in MEK162 sensitiveHCT116 and SW620 xenografts.

FIG. 23B shows whole body imaging of FiMEK nanoparticles in A375 andSW620 xenografts 24 h post administration.

FIG. 23C shows percentage % of tumor size change as calculated from day0.

FIG. 23D shows growth inhibition of different regiments.

FIG. 23E shows evaluation of apoptosis after single administration ofMEK162 or FiMEK.

DETAILED DESCRIPTION

It is contemplated that methods of the claimed invention encompassvariations and adaptations developed using information from theembodiments described herein.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where methods aredescribed as having, including, or comprising specific steps, it iscontemplated that, additionally, there are compositions of the presentinvention that consist essentially of, or consist of, the recitedcomponents, and that there are methods according to the presentinvention that consist essentially of, or consist of, the recitedprocessing steps.

It should be understood that the order of steps or order for performingcertain action is immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

The mention herein of any publication, for example, in the Backgroundsection, is not an admission that the publication serves as prior artwith respect to any of the claims presented herein. The Backgroundsection is presented for purposes of clarity and is not meant as adescription of prior art with respect to any claim.

Fucoidan is a sulfated polysaccharide that is found in various speciesof brown algae and brown seaweed. It can be obtained and purified fromnatural sources, or it may be synthesized. In general, fucoidan has anaverage molecular weight of from about 10,000 to about 30,000 (e.g.,about 20,000), but other molecular weights may be found as well.Naturally-occurring fucoidan includes F-fucoidan, which has a highcontent of sulfated esters of fucose (e.g., no less than 95 wt. %), andU-fucoidan, which contains sulfates esters of fucose but is about 20%glucuronic acid. The fucoidan used in various embodiments describedherein contains no less than 50 wt. %, no less than 60 wt. %, no lessthan 70 wt. %, no less than 80 wt. %, no less than 90 wt. %, or no lessthan 95 wt. % sulfate esters of fucose.

FIG. 1 is a schematic diagram 100 illustrating the preparation ofpH-sensitive fucoidan nanogels for the delivery of doxorubicin (FiDOX).The pH sensitivity is conferred by hydrozone linkages betweendoxorubicin and polyethylene glycol (PEG). The fucoidan and DOX-PEG-DOXconstructs are assembled via a layer-by-layer approach. Fucoidan (Fi) at102 is contacted with the DOX-PEG-DOX construct (DPD) at 104 in thepresence of a phosphonobile salt (PBS), thereby forming hydrogelaggregates at 106. The resulting aggregates are sonicated to form FiDOXnanoparticles. In one example, the particles had average diameter offrom about 150 nm to about 170 nm, with a zeta potential of −55 mV.

In various embodiments, the average particle diameter of FiDOX, or otherdrug-containing fucoidan nanogel, is from about 20 nm to about 400 nm,or from about 100 nm to about 200 nm, or from about 150 nm to about 170nm. The average particle diameter may be measured, for example, viadynamic light scattering (DLS) of a nanogel dispersed in a solvent, orcan be measured via transmission electron micrograph (TEM). In someembodiments, the nanogel has a substantially monodisperse particle size(e.g., has polydispersity index, Mw/Mn of less than 20, more preferablyless than 10, and still more preferably less than 5, less than 2, orless than 1.5, e.g., has polydispersity index in the range from 0 to 1,e.g., from 0.05 to 0.3). Nanoparticles similar to FiDOX can besynthesized to encapsulate the drug vincristine, or other cationicdrugs, by replacing the DOX-PEG-DOX construct with another drugconstruct containing the desired drug.

FIG. 2A shows vials containing fucoidan-paclitaxel nanoparticles(FiPXL). These were prepared using a self-assembly approach, withoutchemical conjugation. This can be performed to encapsulate other drugsas well, such as ispinesib, MEK162, and sorafenib, for example. FIG. 2Bshows the chemical structure of paclitaxel (PX) and fucoidan (Fi) in thefucoidan-paclitaxel nanoparticles.

FIG. 3A shows plots of dynamic light scattering measurements of FiDOXnanogels, showing the particle diameter characterization is stable overat least seven days. FIG. 3B shows transmission electron microscopeimages of the FiDOX nanogels at different concentrations andmagnification.

FIG. 4 is a graph showing the percentage of released doxorubicin fromFiDOX nanogels over time, as a function of pH. Low pH allows fasterrelease due to the breakage of hydrazone bonds.

FIG. 5A shows a plot of fluorescence intensity demonstrating in vitroactivity of FiDOX nanogels. The binding of FiDOX to immobilizedP-selectin was estimated by measuring fluorescence intensity of boundparticles. Soluble fucoidan was able to inhibit binding. A recombinanthuman P-selectin protein was immobilized on an ELISA plate. FiDOXparticles were added to the wells for 15 min and then washed. The boundparticles were detected with a fluorescence plate reader. Free fucoidanwas used to inhibit the binding of the particles to the immobilizedP-selectin on the surface. The particles did not bind to immobilizedalbumin (BSA).

FIG. 5B shows a plot of an MTT cell viability assay. The plot shows thatFiDOX was more cytotoxic to B16F10 cells compared to polyglutamicacid-based nanogels.

FIG. 6 shows bioluminescence images at day 21 of testing, demonstratinganti-tumor efficacy of FiDOX nanogels. A luciferase-expressing B16F10melanoma lung metastasis model was used. The cells were injected intothe tail vein at day 0. The FiDOX particles and controls were injectedat day 7. The progression of metastasis was monitored withbioluminescence imaging after injection of luciferin. Thebioluminescence images show the luciferase-expressing B16F10 cancercells after injection of D-luciferin, 21 days after inoculation and 14days after a single treatment. The FiDOX nanoparticles at 30 mg/kg andabove clearly show more effective treatment than the untreatedspecimens, as well as specimens administered free DOX drug (not innanoparticle form), or fucoidan nanoparticles without the DOX drug (FiNPs).

FIG. 7 is a plot of bioluminescence from the same test, showinganti-tumor efficacy of FiDOX nanogels. Here in FIG. 7, the median numberof photons/sec/cm²/steradian was measured at given time points todemonstrate decreased tumor burden in FiDOX treated mice.

FIG. 8 is a plot showing laboratory mouse survival curve data in theB16F10 melanoma lung metastasis model treated with a single injection ofFiDOX nanoparticles, injected on day 7. The results compared favorablyto an injection of free doxorubicin (DOX), fucoidan alone (Fi), and theuntreated control.

FIG. 9 is a schematic showing fucoidan-ispinesib nanogels (Fi-ISP) andanalogous nanoparticles made by combining ispinesib with fucoidan orPoly Glutamic Acid (PGA) or PGA-PEG. Nanoparticles were formed bynon-covalent assembly. Dynamic light scattering (DLS) plots are shown at906, 910, and 914. FIGS. 10A and 10B are electron micrographs of thefucoidan-ispenesib nanoparticles (Fi-ISP) and PGA-ispinesibnanoparticles. FIG. 11 is an electron micrograph of fucoidan-MEK162nanoparticles.

1 mg of MEK162 in 0.1 ml of DMSO was added dropwise to 15 mg of Fucoidanin 0.5 ml of sodium bicarbonate. The mixture was immediately sonicatedfor 2 min with a probe sonicator (40%) under ice. The mixture wascentrifuged at 20,00 g for 20 min and the pellet was re-suspended in 1ml PBS containing 1 mg of Fucoidan and was again sonicated for 2 minunder ice. The particles were characterized with DLS, TEM and zetapotential measurements. 155 nm particles were obtained with −50 mVsurface zeta potential.

Experimental Examples

Preparation of DOX-PEG-DOX (DPD):

10 mg of Hydrazide-PEG-hydrazide, NH2NH-PEG-NHNH2, MW 3400 (from NANOCS)and 10 mg Doxorubicin were dissolved in 3 ml methanol containing 1004,of glacial acetic acid. The mixture was stirred in the dark for 24 h andthen slowly precipitated in cold acetone/ether (2:1), collected withcentrifugation (15,000 g, 20 min) and dried with vacuum. The product,DOX-PEG-DOX (DPD) was purified with Sephadex G25 PD10 desalting columnwith water as eluent and then lyophilized.

Preparation of FiDOX and DexDOX Nanoparticles:

Fucoidan from Fucus vesiculosus (SIGMA) and DPD were both dissolved indouble distilled water and were mixed together at a weight ratio of 1:1and formed immediate gel aggregates. The aggregates were collected withcentrifugation (15,000 g 10 min) and re-suspended in PBS containingexcess of ×5 Fucoidan. The mixture was sonicated with a probe sonicator40% intensity (sonics vibra-cell) for 10 sec until a clear dark redsolution appeared containing nanoparticles. The particles were collectedwith centrifugation (30,000 g 30 min), re-suspended in PBS, andsonicated in a bath sonicator for 10 min. The particles werecharacterized with DLS, TEM, and zeta potential measurement, and 150 nmparticles were obtained with −55 mV surface zeta potential measurements(FIGS. 20A-B). FIG. 20C shows SEM images of FiDOX nanoparticles. Scalebar is 100 nm. FIG. 20D shows that the sizes of FiDOX stay constant overa 5 day period. FIG. 20E shows the release of DOX over time for pH 7.4and pH 5.5. FIG. 20E shows release of PXL over time for pH 7.4 and pH5.5.

Preparation of FiPAX and DexPAX Nanoparticles:

Paclitaxel-encapsulated fucoidan/dextran sulfate nanoparticles (FiPAXand DexPAX) were synthesized using a nano-precipitation method. 0.1 mlof paclitaxel dissolved in DMSO (10 mg/ml), was added drop-wise (204,per 15 sec) to a 0.6 ml aqueous polysaccharide solution (15 mg/ml)containing IR783 (1 mg/ml) and 0.05 mM sodium bicarbonate. The solutionwas centrifuged twice (20,000 G 30 min) and re-suspended in 1 ml ofsterile PBS. The suspension of nanoparticles was sonicated for 10 secwith a probe sonicator at 40% intensity (Sonics). The resultednanoparticles had zeta potential of −52 mV and a size of 95 nm with aPDI of 0.12 (FIGS. 20A-B). By suspending the nanoparticles in lowervolumes, it was possible to solubilize Paclitaxel (PXL) up to 16 mg/mlin saline solution, which is 2000 times better than free drug. Thenanoparticles were lyophilized with a saline/sucrose 5% solution andreconstituted in water at this concentration. FIG. 20C shows SEM imagesof FiPAX nanoparticles. Scale bar is 100 nm. FIG. 20D shows that thesizes of FiPAX stay constant over a 5 day period. FIG. 20E shows therelease of DOX over time for pH 7.4 and pH 5.5. FIG. 20E shows releaseof PXL over time for pH 7.4 and pH 5.5.

Preparation of Fucoidan+Albumin Nanoparticles Containing Sorafenib:

1 mg of Sorafenib (LC labs) in DMSO was added to 4 mg of Human SerumAlbumin (HSA, Sigma) in 0.3 ml of PBS (pH 4 acidified with HCl) to forma milky white mixture. 3 mg of Fucoidan in 0.3 ml water was added to themixture. The mixture was bath sonicated for 2 min and 0.3 ml of sodiumbicarbonate 100 mM was added until pH 8 was reached. The mixture wassonicated with a probe sonicator for 20 sec under ice and white clearsolution containing nanoparticles. The solution was centrifuged at 30,00g for 20 min and the pellet was re-suspended in PBS followed by bathsonication. 90 nm particles were obtained with −42 mV surface zetapotential (FIGS. 20A-B).

Preparation of Fucoidan Nanoparticles Containing Paclitaxel:

1 mg of Paclitaxel in 0.1 ml of ethanol was added dropwise to 5 mg ofFucoidan in 0.5 ml of water. The mixture was immediately sonicated for 2min with a probe sonicator (40%) under ice. The mixture was centrifugedat 20,00 g for 20 min and the pellet was re-suspended in 1 ml PBScontaining 1 mg of Fucoidan and was again sonicated for 2 min under ice.The particles were characterized with DLS, TEM and zeta potentialmeasurements (FIGS. 20A-B). 180 nm particles were obtained with −51 mVsurface zeta potential.

While the invention has been particularly shown and described withreference to specific preferred embodiments, it should be understood bythose skilled in the art that various changes in form and detail may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

Preparation of Fucoidan-Albumin Nanoparticles Containing Paclitaxel andNear IR Dye: Conjugation of Fucoidan to BSA Via Mallard Reaction:

150 μl of BSA (20 mg/ml) was mixed with 150 μl of Fucoidan solution (80mg/ml), then 150 μl of 0.1 M sodium bicarbonate buffer, pH 8.0, wasadded. The mixture was frozen at −80° C., freeze-dried, and heated at60° C. for 5 hr. After heating, samples were dissolved in 1 ml of water,and purified with Sephadex G25 PD10 column to remove salts and unboundsugar, then freeze dried.

Preparation of Particles from Fucoidan Conjugated BSA:

The Fucoidan BSA conjugate (Fi-BSA, 15 mg) was dissolved in 0.5 ml ofwater. 0.1 mg of IR783 (Sigma) in water was added to the solution. 1 mgof Paclitaxel in 0.1 ml of ethanol was added dropwise and the mixturewas sonicated with a probe sonicator for 1 min. The mixture wascentrifuged at 20,00 g for 20 min and the pellet was re-suspended in 1ml PBS. 110 nm particles were obtained with −45 mV surface zetapotential.

Binding of Nanoparticles to Immobilized P-Selectin:

Human recombinant P- and E-selectin (50 ng in 50 μl) was added to highhydrophobicity 96 well elisa plate and incubated at 4° C. overnight. Thewells were washed with PBS, incubated with BSA (3% 0.2 ml), andincubated with FiPAX or DexPAX in Hank's balanced salt solution (HBSS)for 15 min. The wells were gently washed three times with HBSS and thebinding of nanoparticles was evaluated using scanning fluorescenceintensity performed by TECAN T2000 (‘multiple reads per well’ mode, ex780 nm, em 820 nm).

Binding of Nanoparticles to P-Selectin Expressing Endothelial Cells:

To induce P-selectin expression, monolayers of bEnd3 cells in 24 wellplates were pre-incubated with TNF-α (50 ng/ml) for 20 min prior to theonset of experiments. Control cells were left untreated. The cells werethen incubated with 20 m/ml of nanoparticle for 45 min and another 15min with CellMask Green (Life Technologies) for membrane staining andHOESCHT 66XX for nuclear staining. The cells were then washed twice withPBS. Images were acquired with an inverted Olympus XX fluorescentmicroscope, equipped with XM10IR Olympus camera with an IR range andEXCITE Xenon lamp. Similar exposure time and excitation intensity wereapplied throughout all experiments. Merged images were obtained viaprocessing with ImageJ. Green—Cell membrane (ex 488 nm, em 525 nm),Blue—Nucleus (ex 350 nm, em 460 nm), Red—IR783 dye in particles (ex 780nm, em 820 nm).

Evaluation of Penetration Through Endothelial and Epithelial Barriers:

A modified Transwell assay was used to test penetration of particlesthrough a monolayer of endothelial cells expressing P-selectin.

bEnd3 cells (5×10⁴ in 0.5 ml) were grown on Transwell inserts in 24wells plate for 7 days. The medium was replaced every other day. Theconfluence of the monolayer was validated with imaging of membrane cellstaining to validate the lack of gaps between cell junctions. Followingactivation by TNF-α as described above, the cells were incubated with 20μg/ml of nanoparticles for 1 h and then samples from the upper chamber(50 μl) and fluorescent intensity was measured with a fluorescence platereader (TECAN T2000) at ex 780 nm, em 820 nm. To visualize the particlesin the endothelial cells on the insert component of the chamber, thecells were washed twice with PBS and then incubated in HBSS. Images wereacquired and processed as described above.

Cell Viability Assay:

bEnd3 cells (5×10⁴) were seeded in a 96-well plate. Nanoparticles wereadded to cells that were pre-activated by TNF-α for 30 min, atequivalent drug concentration, and were incubated for 1 h at 37° C.Cells not activated with TNF-α were treated similarly. The drug solutionwas then removed and replaced with fresh medium, followed by 72 h ofincubation at 37° C. Cell survival was assayed by discarding the mediumand adding 100 μl of fresh medium and 25 μl of 5 mg/ml MTT solution inPBS to each well. After 90 minutes, the solution was removed and 200 μlof DMSO were 10 added. Cell viability was evaluated by measuring theabsorbance of each well at 570 nm relative to control wells.

Anti-Tumor Efficacy in Bilateral s.c Model of 3LL:

Murine Lewis lung carcinoma (LLC) were maintained in Dulbecco's ModifiedEagle Medium (DMEM) cell culture medium supplemented with 10% fetalbovine serum, 1 mM Na pyruvate, and 50 ug/ml penicillin andstreptomycin. Tumor cells were subcutaneously implanted (1×10⁶ cells perinjection) in both hind limbs of eightweek old hairless SKH-1 mice. Thetumor models were used for biodistribution and tumor growth studies whenthe tumor size reached 0.5 cm in diameter.

Irradiation of the tumors was conducted at 6 gy doses using X-rayirradiator.

Near Infrared Imaging In Vivo:

Four hours after irradiation, 200 μl (1 mg/ml) of the nanoparticleslabeled with IR783 were injected via the tail vein. Biodistribution ofthe particles within the tumor-bearing mice was monitored with nearinfrared (NIR) imaging. NIR images were taken with an IVIS imagingsystem at various time points. Radiance (photons/sec/cm²) was measuredwithin the tumor region (region of interest, ROI) using the programLivingImage 4.2 provided by Xenogen.

Inhibition of Tumor Growth and Lung Metastasis of B16-F10 Melanoma:

C57BL/6 mice were inoculated intravenously (i.v.) with 1×10⁵B16-F10cells on day 0 and the tumor was allowed to establish until day 7. Inone experiment, mice were divided randomly into 5 groups and injectedi.v. with FiDOX, Fi, DexDOX.

After treatment, mice were monitored up to 8 or 17 weeks, depending onthe treatment received. At the end of the experiments, mice weresacrificed, their lungs were collected, and the number ofsurface-visible tumors was examined. The Kaplan-Meier method was used toevaluate survival.

Establishment of Tumor Xenografts and Studies in Nude Mice:

Six-week-old female athymic NU/NU nude mice were injected subcutaneouslywith 5×10⁵ of A375, SW620, LOVO, and HCT116 in 100 ml culturemedia/Matrigel at a 1:5 ratio. For cell-line-derived xenografts, animalswere randomized at a tumor volume of 70 to 120 mm³ to four to sixgroups, with n=8-10 tumors per group. Animals were orally treated dailywith MEK162 (10 mg/kg or 30 mg/kg in 0.5% carboxymethylcellulose sodiumsalt [CMC]; Sigma). Xenografts were measured with digital caliper, andtumor volumes were determined with the formula: (length× width)× (π/6).Animals were euthanized using CO₂ inhalation. Tumor volumes are plottedas means±SEM. Mice were housed in air-filtered laminar flow cabinetswith a 12-hr light/dark cycle and food and water ad libitum.

Immunohistochemistry (IHC):

For xenograft samples, dissected tissues were fixed after (e.g.,immediately after) removal in a 10% buffered formalin solution for amaximum of 24 h at room temperature before being dehydrated and paraffinembedded under vacuum. The tissue sections were deparaffinized withEZPrep buffer, antigen retrieval was performed with CC1 buffer, andsections were blocked for 30 minutes with Background Buster solution(Innovex). Human P-Selectin/CD62P Monoclonal Antibody (Catalog #BBA30)at 5-15 μg/mL overnight at 4° C. Other antibodies (CD31, P-selectin IFC,Tunel and Cle-PARP) were applied and sections were incubated for 5 hr,followed by a 60 minute incubation with biotinylated goat anti-rabbitIgG (Vector labs, cat#PK6101) at a 1:200 dilution.

As described herein, it has been identified that human tumors (e.g.,lymphomas) express P-selectin primarily on cancer cells and to a lesserextent in the vasculature. Because of the augmented expression oncertain tumor cells and vasculature, P-selectin was tested on targetednanoparticles in a murine model that express P-selectin in both cancerand endothelial cells, models that only express endothelial P-selectin,and models that do not express P-selectin but it can be induced byradiation. For each of the models, appropriate drugs were chosen toachieve high response to a single injection, which demonstrated theplatform capabilities of Fi-based nanoparticles.

There was a significant increase in fucodian particle accumulation inP-selectin expressing tumors on cancer cells and endothelial cells (PDXand irradiated 3LL) in tumor bearing mice. An active mechanism ofdelivery of the chemotherapeutic agents loaded fucoidan nanoparticles(FiDOX and FiPAX) in P-selectin positive aggressive lung metastases andPDX models was not seen in the control nanoparticles with similar chargeand size (DexDOX and DexPax. To further characterize thepharmacodynamics of fucoidan based particles, the activities of areversible kinase inhibitor were investigated. The use of a reversibleMEK inhibitor encapsulated in fucoidan nanoparticles allowed evaluationof kinase inhibition in cancer cells and correlation with drug deliveryto cancer cells. Comparison of a clinically relevant regimen of dailyadministration of MEK162 to a single or weekly dose of the nanoparticleformulation was performed. A single or a weekly administration of areversible inhibitor such as MEK162 encapsulated in a nanoparticle wassimilar to or more effective as a daily administration. Thisdemonstrates the effectiveness of the delivery system to reach not justendothelial cells but also cancer cells. The reduction of a chronic andsystemic inhibition of the pathway and the increase in local tumorconcentrations for prolonged periods of time using Fi nanoparticles willbe more efficacious and better tolerated.

Because the overexpression of P-selectin on endothelial cells and cancercells varies substantially from patient to patient, radiation wasexamined as a way to induce P-selectin locally. In tumors withoutP-selectin expression, it was demonstrated that radiation increasesendothelial P-selectin levels as well as particle accumulation andanti-tumor efficacy. The ability to ‘turn on’ expression of andtranslocation of P-selectin using radiation has a unique advantage sinceit could render virtually any tumor vulnerable to P-selectin targetedsystems. Also, unexpectedly, non-irradiated tumors experienced asignificant therapeutic benefit by a mechanism which may be akin to theabscopal effect.

P-selectin was investigated as a target for localized drug delivery totumor sites, including metastases. It was found that many human tumorssurprisingly express P-selectin spontaneously within their stroma, tumorcells, and tumor vasculature. A nanoparticle carrier was synthesized forchemotherapeutic and targeted therapies using the algae-derivedpolysaccharide, fucoidan, which exhibits nanomolar affinity forP-selectin. It was found that the targeting of activated endotheliumimproved the penetration of fucoidan-based nanoparticles throughendothelial barriers, leading to a therapeutic advantage inP-selectin-expressing tumors and metastases. The encapsulation of bothchemotherapeutic drugs and a reversible MEK inhibitor conferred atherapeutic benefit in P-selectin-expressing tumors, suggesting improveddelivery to tumor tissue. On exposing tumors to ionizing radiation,which induced expression of P-selectin, a significant increase innanoparticle localization and anti-tumor efficacy in tumors that do notspontaneously express the target was observed.

Expression of P-Selectin in Human Cancers:

In order to determine the prevalence of P-selectin expression in cancertissues, ˜400 clinical samples were assessed via immunohistochemistry.(Table Si). As shown in FIGS. 12A and 12B, it was found that P-selectinis highly expressed within multiple types of tumors and theirmetastases, including human lung (19%), ovarian (68%), lymphoma (78%)and breast (49%). Abundant expression of P-selectin was found in thestroma and vasculature surrounding the tumor cells. However in a subsetof cancers, expression of P-selectin on tumor cells was observed.Moreover, significant genomic alterations to the P-selectin gene (SELP)were noted in The Cancer Genome Atlas (TCGA) (FIG. 12C). It was foundthat SELP is amplified in many cancers including breast (27.5%), liver(15%), bladder urothelial carcinoma (13.4%), and lung adenocarcinoma(12.2%). Moreover, the expression of SELP is associated with poorprognosis in squamous cell carcinoma of the lung and renal cellcarcinoma. (FIG. 12C). The abundant expression of P-selectin in cancerprompted the development a P-selectin-targeted vehicle for selectivedrug delivery.

P-Selectin Mediated Transport of Nanoparticles:

To design a P-selectin targeted drug delivery system, fucoidan(Fi)-based nanoparticles were prepared to encapsulate three differentdrug classes with dose-limiting toxicities. Fucoidan-encapsulatedpaclitaxel (PAX) nanoparticles (FiPAX) were synthesized byco-encapsulating paclitaxel, and a near infra-red dye (IR783) tofacilitate imaging, via nano-precipitation as described above inPreparation of FiPAX and DexPAX nanoparticles (FIG. 12D). A reversibleMEK inhibitor, MEK162 was encapsulated in fucodian nanoparticles (FiMEK)in the same manner that FiPAX was prepared. Fucoidan-encapsulateddoxorubicin (DOX) nanoparticles (FiDOX) were synthesized vialayer-by-layer assembly of a cationic doxorubicin-polymer conjugate viapH sensitive hydrazone bond (DOX-PEG-DOX, DPD) and the anionic fucoidan(FIG. 12E). The DPD conjugate was synthesized via pH-cleavable hydrazinelinkages to promote release of the drug in the acidic tumormicroenvironment or lysosomes. The FiDOX, FiPAX and FiMEK nanoparticlesmeasured 150±8.1, 105±4.2 and 85±3.6 nm in diameter respectively, andexhibited approximately −55 mV surface charge (zeta potential).Microscopy showed relatively uniform spherical morphology. As shown inTable 1 below and in FIG. 13, the particles exhibited good serumstability and reconstituted after lyophilization.

TABLE 1 Parameters (units) Control (NT) FiDox (24 Hrs) FiPax (24 Hrs)WBCs (K/μL)  6.90 ± 0.91  4.71 ± 0.28 5.21 ± 0.70 NE (K/μL)  3.71 ± 3.23 1.46 ± 0.12 1.61 ± 0.48 LY (K/μL)  3.03 ± 2.41  3.20 ± 0.29 3.51 ± 0.26MO (K/μL)  0.15 ± 0.05  0.06 ± 0.03 0.07 ± 0.02 EO (K/μL)  0.03 ± 0.02 0.01 ± 0.01 0.01 ± 0.01 BA (K/μL)  0.00 ± 0.00  0.00 ± 0.00 0.00 ± 0.00RBC (M/μL) 10.30 ± 0.69 10.55 ± 0.60 10.55 ± 0.54  Hb (g/dL) 13.30 ±0.99 13.87 ± 0.50 13.70 ± 0.52  HCT (%) 45.05 ± 2.05 45.73 ± 1.96 45.30± 2.78  MCV (fL) 43.75 ± 0.92 43.40 ± 1.14 42.90 ± 0.95  MCH (pg) 12.90± 0.14 13.20 ± 0.61 13.00 ± 0.40  MCHC (g/dL) 29.50 ± 0.85 30.40 ± 0.6930.30 ± 0.89  PLT (K/μL) 1082.00 ± 203.65 753.33 ± 50.08 890.33 ± 125.92

To assess the selectivity of nanoparticle targeting to P-selectin, anuntargeted control drug-loaded nanoparticle lacking the fucoidancomponent was synthesized. Dextran sulfate-encapsulated paclitaxel(DexPAX) nanoparticles were assembled with the same methods as usedabove. The binding of FiPAX and DexPAX was compared to immobilized humanrecombinant P-selectin, E-selectin, and BSA, thereby confirming theselective binding to P-selectin in a dose dependent manner (FIG. 12E:P<0.05).

It was investigated whether a fucoidan-based nanoparticle would bind toactivated endothelium and translocate the endothelial barrier. Theability of fucoidan nanoparticles to penetrate through endothelium andinto tumor tissue was assessed using a modified Transwell assay. Murinebrain endothelial (bEnd.3) cells were grown on the top chamber'smembrane, and P-selectin expressing tumor spheroids were grown in thebottom chamber (FIG. 14A). The penetration of the nanoparticles throughthe bEnd.3 monolayer upon activation by TNF-α was measured. The quantityof FiPAX nanoparticles recovered from the bottom chamber increasedsignificantly by ˜30% (FIG. 14B) in the presence of TNF-α, while DexPAXincreased by 15%, suggesting that endothelial activation enhanced thetranslocation of the FiPAX nanoparticles. The FiPAX nanoparticles weretaken up by the endothelial cells only upon activating with TNF-α, andthe cells did not take up the control DexPAX nanoparticle in either case(FIG. 14C). Penetration of the nanoparticles into tumor spheres viafluorescence microscopy was quantified. As shown in FIGS. 2D to 2E, a3-fold increase in the FiPAX-encapsulated dye fluorescence in the tumorspheres upon activation with TNF-α, as well as greater penetration intothe spheres, compared to the DexPAX nanoparticles (FIG. 14d-14e ). Theseobservations suggest that endothelial activation mediates increasedtransport of P-selectin-targeted nanoparticles across an endothelialbarrier and into solid tumor tissue compared to untargeted particles.These findings support that particle extravasation and tumor penetrationto P-selectin expressing tumors in vivo is possible.

Anti-Tumor Efficacy Mediated by P-Selectin:

To determine the net efficacy of P-selectin targeting in vivo, apatient-derived xenograft (PDX) model of SCLC which expresses P-selectinwas used (FIG. 15A). This PDX expressed P-selectin both in tumorendothelium and cancer cells (FIG. 13A: LX36). When tumors reached 70mm³, mice were randomized into 4 arms: PBS, FiPAX, DexPAX and paclitaxel(PAX). Upon 24 h and 72 h after injection of nanoparticles, the micewere imaged to compare particle localization. The average fluorescenceintensity was 2.5 times higher than that of DexPAX after 24 h, and thesignal difference increased to 4.1 times at 72 h (FIG. 15A, FIG. 16B).Upon administration of a single injection of each treatment, FiPAXnanoparticles significantly inhibited tumor progression as compared tofree paclitaxel or untargeted DexPAX nanoparticles (FIG. 15B).

To investigate the radiation-induced expression of P-selectin in a modelthat does not spontaneously express the target, nude mice wereinoculated in both flanks with Lewis lung carcinoma (3LL) cells. Theresulting tumor did not endogenously express P-selectin, as observed bytissue staining (FIG. 15C). The right flank tumor of each mouse wasirradiated with 6 Gy, while the left tumors were left un-irradiated. Itwas observed that the expression of P-selectin in the irradiated tumorwas apparent by 4 hours and increased substantially by 24 hours (FIG.15C). Notably, P-selectin expression was found in the non-irradiatedtumors of the irradiated mice after a 24 hour delay (FIG. 15C), as wellas an increase in soluble P-selectin (sP-selectin) in the blood of theirradiated mice (FIG. 16).

It was investigated whether radiation could selectively guideP-selectin-targeted drug carrier nanoparticles to a tumor site to resultin a net therapeutic benefit. The 3LL bilateral tumor model wasirradiated with 6 Gy on the right tumor before injecting the mice i.v.with nanoparticles 4 hours later. To distinguish the effects ofradiation-induced P-selectin targeting from an EPR effect or non-inducedP-selectin, untargeted DexPAX nanoparticles and non-irradiated controlmice were included. At 24 hours after treatment, the fluorescence signalfrom FiPAX nanoparticles were 3.8 times higher in the irradiated tumorsover non-irradiated tumors, while there was no difference in theDexPAX-treated mice (FIG. 15E, 16D). Growth was halted in tumorsreceiving both radiation and FiPAX nanoparticles, resulting in theircomplete tumor disappearance (FIG. 15F). Notably, in mice treated withFiPAX nanoparticles and radiation, significant inhibition was observedin the non-irradiated tumors, suggesting an abscopal-like effect onanti-tumor efficacy mediated by the nanoparticle. To corroborate the invivo observations, FiPAX binding to radiation-induced P-selectinexpression in vitro were evaluated. In bEnd.3 endothelial cells,radiation-mediated P-selectin expression was observed in adose-dependent manner. The irradiated cells took up FiPAX nanoparticles,while little uptake of DexPAX nanoparticles was measured (FIGS. 16A and16B: P<0.05).

Anti-Tumor Efficacy in Endogenous P-Selectin Expressing Metastases:

The anti-tumor efficacy of P-selectin-targeted drug carriernanoparticles was assessed against an aggressive experimental metastasismodel. The i.v. injection of 10⁵ B16F10 melanoma cells results in lungmetastases which exhibit P-selectin expression in the associatedvasculature (FIG. 17A-B). Three different doses of FiDOX(fucoidan-encapsulated doxorubicin) nanoparticles were then administeredto identify a therapeutic window. The mice were divided into 6 groups of5 mice and treated with a single dose of either free doxorubicin at 6mg/kg or 8 mg/kg, close to the maximum tolerated dose, fucoidan (30mg/kg), as a vehicle control, and three concentrations of FiDOX (1mg/kg, 5 mg/kg and 30 mg/kg). The treatment with FiDOX nanoparticles atall three concentrations resulted in decreased tumor burden andprolonged survival upon a single injection, whereas an equivalent amountof free doxorubicin at its maximum tolerated dose, did not have asignificant effect (FIG. 17C). Fucoidan alone also showed no survivalbenefit. After 7 days post-treatment, tumor bioluminescence shows aclear reduction in median photon count in the medium and the high dosegroups (FIGS. 17D, 17E). Signs of toxicity as measured by weight loss orcomplete blood count were not observed FIG. 18). The anti-tumor efficacyof FiDOX nanoparticles was also compared to the untargeted DexDOXnanoparticle control and untargeted drug-polymer conjugate, DOX-PEG-DOXat equivalent doxorubicin doses of 8 mg/kg. The mean survival of theFiDOX group was significantly higher at 68.8 days with 40% cured micecompared to DexDOX at 40.2 days with no cures, DOX-PEG-DOX at 39.2 days,and untreated 32.4 at days (FIG. 17B, p=0.005).

P-Selecting Targeting of Mechanistically Targeted Drugs:

The Ras-ERK pathway is frequently hyperactive in substantial types ofcancers including melanoma, colorectal, and lung cancers, and thereforesMEK/ERK reversible inhibitors have been tested in large number ofclinical trials in RAS- and BRAF-mutated cancers. Blocking this pathwayusing systemic MEK/ERK inhibitors is, however, dose-limiting with onlytemporal target inhibition. At high dosage, these treatments causetoxicity in patients such as severe rash and chronic serous retinoscopy(CSR).

It is described herein how P-selectin-targeted delivery improved theefficacy of reversible kinase inhibitors which are specific to cancercells. For example, the delivery of MEK inhibitor to the tumormicroenvironment using P-selectin targeted nanoparticles increased theconcentration of drug in the tumor itself, therefore prolonging theduration of inhibition and reduce systemic toxicity.

To this end, MEK162 was co-encapsulated with IR783 within fucoidan-basednanoparticles (FiMEK) in the same manner that FiPAX was prepared. Invitro, the release of the MEK162 by the nano-particle was sustained withmaximum of 85% reached in 24 hours and accelerated by acidic pH (FIG.23A). Data shows that free MEK162 and MEK162 loaded fucoidannanoparticles (FI-MEK) had similar biochemical and anti-tumor activityagainst BRAF mutated melanoma (A375), and NRAS mutated lung (A549) andKRAS mutated colorectal (HCT116 and SW620) cancer cells in vitro (FIG.19A, FIG. 21).

In tumor bearing mice, a single administration of FIMEK inducedsignificant tumor growth inhibition compared to no effect of oraltreatment. A375 and SW620 tumor bearing mice were treated with a weeklydose of MEK162, FiMEK and a daily dose of free MEK162. It was observedthat a weekly dose of FiMEK was more effective than a weekly dose offree MEK, and had comparable efficacy with a daily administration. Thisresult was validated in two other models of LOVO and HCT116 xenografts(FIGS. 23A-23E).

To further understand the enhanced efficacy of FiMEK compared to oralMEK162, the pharmacodynamics were assessed by measuring the levels ofpERK and cleavage of PARP on tumors treated with MEK162 or FiMEK at 2 hand 16 h after administration (FIG. 19C). The data shows a similarinhibition of pERK after 2 hours of treatment. However, significantprolong of pERK inhibition was observed in mice treated with FIMEK whencompared to mice treated with oral MEK162. An association betweenprolong inhibition of ERK and induction of apoptosis was observed,indicating the importance of the duration of pathway inhibition.Immunohistochemistry of Clevage PARP on xenogfrat HCT116 tumors treatedwith MEK162 or MEK-IR was assessed to confirm the death of tumor cells(FIGS. 19D, 19E). FIG. 22 shows the drug release profile MEK162 fromnanoparticles over time at different pH.

What is claimed is:
 1. A polymeric nanoparticle with affinity toP-selectin, the nanoparticle comprising a non-covalent assembly of asulfated polymer species comprising free hydroxyl moieties and freesulfate moieties capable of targeting P-selectin, the sulfated polymerspecies comprising one or more of a sulfated polysaccharide, a protein,or a fucoidan; a hydrophobic drug; and a dye comprising a fluorophore;wherein the nanoparticle has an intensity-weighted average diameter asdetermined by dynamic light scattering of about 20 nm and about 200 nm;and the non-covalent assembly is a self-assembly of the sulfated polymerspecies around the hydrophobic drug.
 2. The nanoparticle of claim 1,wherein the sulfated polymer species comprises a fucoidan.
 3. Thenanoparticle of claim 1, wherein the hydrophobic drug comprises a memberselected from the group consisting of paclitaxel, MEK162, docetaxel,Camptothecin, sorafenib, ispinesib, LY294002, Selumetinib, PD184352,5-fluorouracil, Cyclophosphamide, Atorvastatin, Lovastatin, etoposide,dexamethasone, gemcitabine, Rapamycin (Sirolimus), and methotrexate. 4.The nanoparticle of claim 1, wherein the nanoparticle has anintensity-weighted average diameter as determined by dynamic lightscattering of about 100 nm to about 200 nm.
 5. The nanoparticle of claim1, wherein the nanoparticle has an intensity-weighted average diameteras determined by dynamic light scattering of about 150 nm to about 170nm.
 6. The nanoparticle of claim 1, wherein the dye is a near infra-reddye.
 7. The nanoparticle of claim 1, wherein the dye is IR783.
 8. Thenanoparticle of claim 1, wherein the nanoparticle is negatively charged.9. A method for manufacturing the nanoparticle of claim 1, the methodcomprising: contacting the hydrophobic drug to the sulfated polymerspecies to form a mixture; and agitating the mixture to formnanoparticles.
 10. The method of claim 9, wherein agitating the mixturecomprises sonicating the mixture.
 11. The method of claim 9, wherein thehydrophobic drug and the sulfated polymer species are precipitatedtogether.
 12. The method of claim 9, wherein the hydrophobic drug andthe sulfated polymer species are precipitated together vianano-precipitation.
 13. A method of treating a subject suffering from aP-selectin associated disease, the method comprising administering tothe subject a formulation comprising the nanoparticle of claim
 1. 14.The method of claim 13, wherein the subject is human.
 15. The method ofclaim 13, wherein the P-selectin associated disease comprises a memberselected from the group consisting of carcinoma, sarcoma, lymphoma,leukemia, sickle cell disease, arterial thrombosis, rheumatoidarthritis, ischemia, and reperfusion.
 16. The method of claim 13,wherein the P-selectin associated disease comprises a tumor.
 17. Themethod of claim 13, the method further comprising administeringradiation to a site in the subject comprising the P-selectin associateddisease.
 18. The method of claim 17, the radiation comprising ionizingradiation.
 19. The method of claim 17, wherein the P-selectin associateddisease comprises a member selected from the group consisting ofcarcinoma, sarcoma, lymphoma, leukemia, sickle cell disease, arterialthrombosis, rheumatoid arthritis, ischemia, and reperfusion.
 20. Themethod of claim 17, wherein the P-selectin associated disease comprisesa tumor.